Cell Fractionation: Separating Cell Organelles Step by Step

Cell fractionation is a laboratory method of breaking cells open and separating their organelles by size and density using centrifugation. It allows scientists to study the structure and chemical activity of each organelle in isolation.

It matters because whole-cell extracts mix all reactions together. By isolating nuclei, mitochondria, ribosomes and other fractions, researchers can assign specific functions and enzymes to specific organelles.

Key Concepts

• Homogenisation: gentle breaking of cells in ice-cold isotonic buffer to release organelles intact.
• Differential centrifugation: spinning at increasing speeds to pellet larger and denser organelles first.
• Density gradient centrifugation: separating organelles through a sucrose gradient by density.
• Pellet: material sedimented at the bottom of the centrifuge tube.
• Supernatant: liquid remaining above the pellet containing smaller components.

Low temperature and isotonic medium prevent organelle damage and enzyme inactivation during the process.

How It Works

1. Tissue is minced and homogenised in cold sucrose buffer to break plasma membranes.
2. The homogenate is centrifuged at low speed (about 600 g) to pellet nuclei and unbroken cells.
3. The supernatant is spun at medium speed (about 10,000 g) to pellet mitochondria, lysosomes and peroxisomes.
4. Higher speed (about 100,000 g) pellets microsomes and ribosomes, leaving soluble cytosol as final supernatant.

Each fraction is then tested for marker enzymes to confirm purity.

Worked Example

A liver homogenate is processed stepwise. Starting with 10 grams of tissue, low-speed centrifugation yields a nuclear pellet containing most DNA. The supernatant spun at 10,000 g gives a mitochondrial pellet; testing shows high activity of succinate dehydrogenase, a mitochondrial marker. A final ultracentrifugation at 100,000 g for 60 minutes pellets ribosomes, and the remaining supernatant contains soluble enzymes like lactate dehydrogenase. If mitochondria originally made up about 20 percent of cell protein, recovering roughly that proportion in the pellet confirms efficient separation without major loss.

Common Misconceptions

• Misconception: Higher speed pellets larger organelles. Correction: larger and denser organelles pellet at lower speeds; smaller ones need higher speeds.
• Misconception: Fractions are perfectly pure after one spin. Correction: fractions overlap and require marker analysis and sometimes gradient purification.
• Misconception: Homogenisation can be done at room temperature. Correction: cold conditions are essential to slow enzyme degradation and preserve organelles.

Real-World Applications

Cell fractionation established that mitochondria perform respiration, ribosomes synthesise proteins and lysosomes contain digestive enzymes. It is used in drug metabolism studies with liver microsomes, proteomics of organelle fractions and quality control of subcellular preparations in biotechnology.

In short, cell fractionation breaks cells gently and uses stepwise centrifugation to isolate organelles. This separation by size and density remains a foundation for understanding cell function.